Novel hyaluronan hydrolase mutant with improved stability and pharmaceutical composition containing the same

Abstract

To provide novel PH20 variants or fragments thereof improved in the stability, enzyme activity and expression level compared to mature wild-type PH20.SOLUTION: The invention provides novel PH20 variants or fragments thereof improved in thermal stability and enzymatic activity of human hyaluronidase, which is an enzyme for hydrolyzing hyaluronic acid, the novel PH20 variants or fragments thereof including one or more amino acid residue substitutions in a variant having a specific amino acid sequence, where additional amino acid residues at the N-terminus and / or the C-terminus are optionally deleted.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a novel human PH20 mutant or a fragment thereof that has improved enzymatic activity and thermal stability of human hyaluronidase, a hyaluronic acid hydrolase. In particular, the present invention relates to a PH20 mutant or a fragment thereof that has the amino acid sequence of SEQ ID NO: 3 and includes substitution, deletion, and / or insertion of one or more amino acid residues, and optionally has a portion of the amino acid residues deleted at the N-terminus and / or C-terminus, a method for producing the mutant, and a pharmaceutical composition containing the mutant. [Background technology]

[0002] Human skin is composed of the epidermis, dermis, and subcutaneous fat layer, and contains six types of glycosaminoglycans: hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate.

[0003] Glycosaminoglycans are composed of repeating disaccharide sugars. The number of linked sugars varies from one glycosaminoglycan to another, ranging from several hundred to several thousand. Among glycosaminoglycans, more than half of hyaluronic acid in the body is found in the skin. Hyaluronic acid is synthesized by hyaluronan synthase present in cell membranes. It exists independently without being bound to proteoglycans and is the only glycosaminoglycan lacking sulfate groups. Other glycosaminoglycans are bound to proteoglycans and contain sulfate groups. Hyaluronic acid is composed of glucuronic acid and N-acetylglucosamine linked together via cross-links of β-1,3 and β-1,4 bonds, with these disaccharides repeating approximately 5,000 times. It is known that approximately one-third (5g) of hyaluronic acid is decomposed in the human body every day.

[0004] Hyaluronidase is an enzyme that breaks down hyaluronic acid located in the extracellular matrix. Six hyaluronidase genes are known to exist in humans (Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1). In humans, Hyal1 and Hyal2 are expressed in most tissues, while PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the cell membrane and acrosome membrane of sperm. However, HyalPS1 is a pseudogene and is not expressed. Hyaluronidases are divided into three types based on the method by which they cleave hyaluronic acid. They are classified into enzymes that cleave the β-1,4 bond between N-acetylglucosamine and glucuronic acid using HO (EC 3.2.1.35) and enzymes that cleave the β-1,3 bond (EC 3.2.1.36), as well as bacterial hyaluronidases that cleave the β-1,4 bond without using HO (EC 4.2.99.1).

[0005] The catalytic amino acid residues of Hyal1 are D129 and E131, and it hydrolyzes hyaluronan through substrate-assisted catalysis. Hyal1 is optimally active at acidic pH 3-4, with no enzymatic activity above pH 4.5. In contrast to Hyal1, PH20 is active over a wide pH range from 3-8.

[0006] Arming et al. (1997) showed that the catalytic amino acids of PH20 are D111 and E113. They designated the first amino acid, Leu, of PH20 without the signal sequence as number 1, so that in PH20 with the signal sequence, the catalytic amino acid residues are D146 and E148, respectively.

[0007] Hyaluronidase hydrolyzes hyaluronic acid, reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing its permeability through tissues (skin). Because the subcutaneous tissue has a neutral pH of approximately 7.0–7.5, PH20 is widely used clinically (Bookbinder et al., 2006). Clinical applications of PH20 include its use as an eye relaxant and an additive to anesthetic injections during ophthalmic surgery, and its coadministration with subcutaneous antibody therapeutics (Bookbinder et al., 2006). Furthermore, given the characteristic of tumor cells overexpressing hyaluronic acid, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the accessibility of anticancer therapeutic agents to tumor cells. It is also used to promote the reabsorption of excess body fluids and blood in tissues.

[0008] PH20 was first identified in guinea pig sperm by Lathrop et al. and is known to be expressed in sperm of various other species. The human PH20 gene was cloned by Lin et al. and Gmachl et al. Human PH20 consists of 509 amino acid residues and has the sequence set forth in SEQ ID NO: 1, which shares 60% amino acid sequence identity with guinea pig PH20. The human PH20 enzyme is encoded by the SPAM1 (sperm adhesion molecule-1) gene and exists on the surface of sperm plasma membrane and the inner surface of the acrosomal membrane, with Ser490 of PH20 bound to GPI (glycosylphosphatidylinositol). Sperm use PH20 to hydrolyze hyaluronan when penetrating the cumulus layer, which is rich in hyaluronan, to enter the egg. PH20 accounts for less than 1% of the protein content in sperm and has six N-glycosylation sites (N82, N166, N235, N254, N368, and N393).

[0009] Currently, the most commonly used commercially available PH20 is extracted from bovine or ovine testicles. For example, Amphadase® (bovine hyaluronidase) ) and Vitrase(R) (ovine hyaluronidase).

[0010] Bovine testicular hyaluronidase (BTH) is a form of wild-type bovine PH20 in which the signal peptide and 56 C-terminal amino acids have been removed during post-translational modification. BTH is also a glycoprotein, with mannose accounting for 5% and glucosamine accounting for 2.2% of the total amino acid content. Repeated administration of animal-derived hyaluronidase to humans in high doses can lead to the production of neutralizing antibodies. Animal-derived hyaluronidase contains other biological substances besides PH20, which may cause allergic reactions when administered to humans (Bookbinder et al., 2006). In particular, the production and use of bovine-derived PH20 is limited due to concerns about mad cow disease. To address these issues, research into recombinant human PH20 protein is underway.

[0011] Recombinant human PH20 protein has been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, animal cells, etc. The recombinant PH20 protein produced in insect cells and yeast differs from human PH20 in the N-glycosylation pattern during the post-translational protein modification process.

[0012] Among hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB IDs: 1FCQ, 1FCU, 1FCV) have been elucidated. Hyal1 consists of two domains: a catalytic domain and an EGF-like domain. The catalytic domain has a (β / α)8 configuration, with eight alpha and eight beta helical repeats, characteristic of the protein's secondary structure (Chao et al., 2007). The EGF-like domain is conserved in all variants of Hyal1 with alternatively spliced ​​C-termini. The amino acid sequences of Hyal1 and PH20 share 35.1% identity, but the protein structure of PH20 has not yet been elucidated.

[0013] Recombinant human PH20 protein has been developed by Halozyme Therapeutic and is sold under the trade name Hylenex (Bookbinder et al., 2006; Frost, 2007).

[0014] Mutation of the catalytic amino acids D146 and E148 of PH20 to asparagine (D146N) and glutamine (E148Q), respectively, resulted in a loss of enzymatic activity (Arming et al., 1997). Furthermore, substitution of R246 with glycine reduced enzymatic activity by 90%, and substitution of E319 with glutamine and R322 with threonine abolished enzymatic activity. A PH20 mutant with 36 amino acids removed from the C-terminus (a truncation from amino acids 474 to 509) exhibited a 75% reduction in enzymatic activity compared to wild-type PH20. This mutant was not secreted extracellularly and persisted in HeLa cells. Deletion of the C-terminal 134 amino acids from PH20 abolished enzymatic activity and prevented extracellular secretion. According to Frost et al., the C-terminal region 477 to 483 of PH20 is essential for soluble expression (Frost, 2007). The activity of the full-length PH20 (1-509) or a mutant truncated at C-terminus 467 was only 10% of that of a mutant truncated at any one of C-terminus positions 477-483 (Frost, 2007).

[0015] Recombinant PH20 is used medically as a delivery agent to promote subcutaneous delivery of pharmaceuticals, to reduce intraocular pressure in patients with ophthalmic diseases, to delay postoperative stenosis, as a dispersion agent to increase the activity of chemotherapeutic agents in diseases such as cancer, and as an adjuvant therapeutic agent for surgical procedures.

[0016] In particular, in recent years, protein pharmaceuticals have been developed as high-concentration, high-volume products with concentrations ranging from tens to hundreds of mg per mL, and recombinant PH20 has been increasingly used as a delivery formulation to promote subcutaneous delivery of such protein pharmaceuticals. In the case of such protein pharmaceuticals, physical stability issues can arise due to increased viscosity and aggregation of the protein at high concentrations. Furthermore, protein aggregation is an irreversible phenomenon, and once a small amount begins to aggregate, aggregation tends to increase further (Schon et al., 2015). Therefore, aggregation in recombinant PH20 administered concomitantly can reduce the stability of the protein pharmaceutical.

[0017] However, existing recombinant PH20 still lacks sufficient thermostability or expression rate. Therefore, there is a great need in the industry for recombinant hyaluronidase with further improved biological and physicochemical properties.

[0018] Arming, S., Strobl, B., Wechselberger, C., and Kreil, G. (1997). In vitro mutagenesis of PH-20 hyaluronidase from human sperm. Eur J Biochem 247, 810-814.

[0019] Bookbinder, L.H., Hofer, A., Haller, M.F., Zepeda, M.L., Keller, G.A., Lim, J.E., Edgington, T.S., Shepard, H.M., Patton, J.S., and Frost, G.I. (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J Control Release 114, 230-241.

[0020] Chao, K.L., Muthukumar, L., and Herzberg, O. (2007). Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 46, 6911-6920.

[0021] Frost, G.I. (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin Drug Deliv 4, 427-440.

[0022] Schoen, A., Clarkson, B.R., Siles, R., Ross, P., Brown, R.K., Freire, E. (2015) Denatured state aggregation parameters derived from concentration dependence of protein stability. Anal Chem 488, 45-50 WO 2020 / 022791A (2020. 1. 30.) Summary of the Invention [Problem to be solved by the invention]

[0023] An object of the present invention is to provide a novel PH20 mutant or fragment thereof that has improved stability, enzymatic activity, and expression rate compared to wild-type PH20, preferably mature wild-type PH20. Another object of the present invention is to provide a composition for treating cancer comprising the PH20 mutant or a fragment thereof, and a method for treating cancer using the same. [Means for solving the problem]

[0024] To achieve the above objectives, the present invention provides a PH20 mutant or fragment thereof that includes a substitution, deletion, and / or insertion of one or more amino acid residues in a hyaluronidase mutant having the amino acid sequence of SEQ ID NO: 3, and optionally, a partial deletion of amino acid residues at the N-terminus and / or C-terminus. The present invention also provides a composition for cancer treatment comprising the above-described PH20 mutant according to the present invention or a fragment thereof, and a treatment method using the same. [Effects of the Invention]

[0025] The PH20 mutant or a fragment thereof of the present invention has an increased protein expression level when expressed in ExpiCHO cells compared to mature wild-type PH20, and the protein aggregation temperature is increased by approximately 4 to 11.5°C, resulting in the effect of being able to be produced efficiently while having high thermal stability.

[0026] Furthermore, the PH20 mutant or fragment thereof of the present invention has improved protein refolding and faster renaturation than mature wild-type PH20, as shown by the results of a substrate-gel assay, which is one of the experiments used to measure hyaluronidase activity, and maintains its original enzymatic activity regardless of the C-terminal cleavage site. Furthermore, the PH20 mutant or fragment thereof of the present invention has low immunogenicity and can be administered repeatedly to the human body. [Brief explanation of the drawings]

[0027] [Figure 1] These figures show the results of SDS-PAGE analysis of various mutants based on the PH20 mutant having the amino acid sequence of SEQ ID NO: 3. The SDS-PAGE analysis results for each mutant were obtained by column chromatography purification using animal cell culture media expressing each mutant, followed by 10% SDS-PAGE analysis of the final purified mutant. Figure 1A shows the SDS-PAGE results for mutants HM98, HM99, HM130, HM143, HM71, HM100, HM131, HM72, HM101, and HM114. Figure 1B shows the SDS-PAGE results for mutants HM63, HM102, HM115, HM64, HM103, HM116, HM125, HM132, HM65, HM133, HM144, HM104, and HM117. Figure 1C shows SDS-PAGE gel results for mutants HM66, HM105, HM134, HM76, HM106, HM135, HM136, and HM67. Figure 1D shows SDS-PAGE gel results for mutants HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM107, HM118, HM90, HM91, HM92, HM93, HM94, and HM95. Figure 1E shows SDS-PAGE gel results for mutants HM126, HM69, HM110, HM138, HM73, HM111, HM121, HM139, HM74, HM112, and HM140. Figure 1F shows the SDS-PAGE results for the HM75, HM141, HM145, HM70, HM77, HM142, HM78, HM79, HM96, HM146, HM147, HM149, and HM150 mutants.

[0028] [Figure 2]This figure shows the expression levels of mature wild-type PH20, and the Hyal2 variant (Hyal2-Variant), Hyal3 variant (Hyal3-Variant), and Hyal4 variant (Hayal4-Variant) in which the M345 to I361 sites of mature wild-type PH20 were replaced with the corresponding sequences of Hyal2, Hyal3, and Hyal4, respectively. SDS-PAGE lane CS is the culture medium sample, lane FT is unbound impurities on the HisTag column, and lane E is the HisTag column eluate.

[0029] [Figure 3]Figure 3A shows the results of SDS-PAGE analysis of various mutants based on the PH20 mutant having the amino acid sequence of SEQ ID NO: 3. Purification was performed by column chromatography using animal cell culture medium expressing each mutant, and the final purified mutants were subjected to 10% SDS-PAGE analysis. Figure 3B shows the results of SDS-PAGE analysis of mutants HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, and HM169. Figure 3B shows SDS-PAGE results for mutants HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, and HM186. Figure 3C shows SDS-PAGE results for mutants HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, and HM205. Figure 3D shows SDS-PAGE results for mutants HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, and HM220. Figure 3E shows SDS-PAGE results for mutants HM231, HM232, HM233, HM234, HM235, HM243, HM245, and HM246. Figure 3F shows SDS-PAGE results for mutants HM254, HM261, HM262, HM263, HM266, HM268, HM271, HM275, HM276, HM279, HM280, HM287, and HM288.

[0030] [Figure 4]The results of SDS-PAGE analysis of the thermal stability of wild-type PH20 (L36-Y482) and the PH20 mutant (F38-F468) having the amino acid sequence of SEQ ID NO: 3 are shown. Lanes A, B, C, and D show the results of SDS-PAGE analysis of the initial wild-type PH20 (lanes A and C) and the PH20 mutant of SEQ ID NO: 3 (lanes B and D) under reduced (lanes A and B) and non-reduced (lanes C and D) conditions, respectively. Lanes E, F, G, and H show the results of SDS-PAGE analysis of the wild-type PH20 (lanes E and G) and the PH20 mutant of SEQ ID NO: 3 (lanes F and H) under reduced (lanes E and F) and non-reduced (lanes G and H) conditions after storage at 42°C for 7 days. DETAILED DESCRIPTION OF THE INVENTION

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0032] In the present invention, when describing each mutant based on wild-type PH20, the positions of amino acid residues follow the positions in the amino acid sequence of SEQ ID NO: 1, and when describing the PH20 mutant based on SEQ ID NO: 3, the positions follow the positions in the amino acid sequence of SEQ ID NO: 3.

[0033] The inventors of the present invention have previously discovered through research that a hyaluronidase PH20 mutant having the amino acid sequence of SEQ ID NO: 1, preferably a mature wild-type PH20, which contains a substitution of one or more amino acid residues in the alpha helix region and / or the region corresponding to the connecting part thereof, preferably the alpha helix 8 region (S347-C381) and / or the connecting part between alpha helix 7 and alpha helix 8 (A333-R346), and optionally has a portion of the N-terminal and / or C-terminal amino acid residues truncated and deleted, has superior efficacy compared to existing wild-type PH20 or fragments thereof, and has filed a patent application (see WO2020 / 022791A).

[0034] In the present invention, "mature wild-type PH20" means a protein consisting of amino acid residues L36 to Y482 or L36 to S490 of SEQ ID NO: 1, in which the signal peptide M1 to T35 and N483 to L509 or A491 to L509, which are not related to the substantial function of PH20, are deleted from the amino acid sequence of wild-type PH20 of SEQ ID NO: 1.

[0035] Specifically, the inventors of the present invention have discovered through previous research that when the amino acid residues corresponding to alpha helix 8 (S347-C381) and / or T341-I361, which are part of the connecting part (A333-R346) between alpha helix 7 and alpha helix 8 in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, are substituted with the corresponding amino acid residues in wild-type Hyal1 having the sequence of SEQ ID NO: 2, expression efficiency and enzymatic activity are improved. Furthermore, they have confirmed that even fragments in which part of the amino acid sequence at the N-terminus and C-terminus is deleted still have excellent expression efficiency and high enzymatic activity.

[0036] [Table 1]

[0037] As a result of continued research, the present researchers have found that a mutant having the sequence of SEQ ID NO: 3, in which the amino acid positions corresponding to T341 to I361 of wild-type PH20 having the amino acid sequence of SEQ ID NO: 1 are replaced with the corresponding amino acid sequence of wild-type Hyal1 having the sequence of SEQ ID NO: 2, contains substitution, deletion, and / or insertion of additional amino acid residues, and further, selectively, even when a portion of the N-terminal and / or C-terminal amino acid residues are deleted, still has excellent expression efficiency and high enzymatic activity, and also has a low protein aggregation temperature (aggregation temperature, T agg ) is significantly improved compared to wild-type PH20, and this discovery led to the completion of the present invention.

[0038] The mutant having the sequence of SEQ ID NO: 3 is a mutant in which 15 amino acid residues, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, have been substituted in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1.

[0039] In this aspect, the PH20 mutant or fragment thereof of the present invention is characterized in that it comprises a substitution, deletion, and / or insertion of one or more amino acid residues in a PH20 mutant having the amino acid sequence of SEQ ID NO: 3, and optionally, a partial deletion of amino acid residues at the N-terminus and / or C-terminus.

[0040] As described above, the mutant having the amino acid sequence of SEQ ID NO: 3 is a mutant in which the amino acid residues T341 to I361 of wild-type PH20 are substituted with the corresponding amino acid residues of wild-type Hyal1 (see Table 2). Existing studies have confirmed that the mutant having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof containing deletions of amino acid residues at the N-terminus and C-terminus has the best activity and stability compared to wild-type PH20.

[0041] [Table 2]

[0042] Specifically, the PH20 mutant or fragment thereof according to the present invention is characterized in that it contains one or more mutations, preferably substitutions, deletions, and / or insertions of amino acid residues, in the sequence of SEQ ID NO: 3, and has a protein aggregation temperature (T agg ) is higher than that of wild-type PH20. Furthermore, the PH20 mutants according to the present invention do not include the wild-type PH20 of SEQ ID NO:1.

[0043] In the present invention, the term "PH20 mutant" is used to refer to a mutant having some amino acid residues mutated in the amino acid sequence of SEQ ID NO: 3, preferably substitution, deletion, and / or insertion of amino acid residues, as well as a mutant having some amino acid residues deleted at the N-terminus or C-terminus in addition to such substitution, deletion, and / or insertion of amino acid residues, and is used in substantially the same sense as the term "PH20 mutant or a fragment thereof."

[0044] Preferably, the PH20 mutant of the present invention is characterized in that, in a mutant having the amino acid sequence of SEQ ID NO: 3, it comprises substitutions, insertions, and / or deletions of amino acid residues at one or more positions selected from the group consisting of R39, D65 to L68, N82, T84, I102 to I105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463, and D461 to V463, and has a protein aggregation temperature (aggregation temperature, T agg ) is improved, i.e., higher, compared to wild-type PH20.

[0045] The PH20 mutant of the present invention may include mutations at 20 or fewer, preferably 17 or fewer, and more preferably 15 or fewer amino acid positions in the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0046] More preferably, the PH20 mutant or fragment thereof according to the present invention has the amino acid sequence of SEQ ID NO: 3 and is selected from the group consisting of R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T, S180A, L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q268I, Q268N, Q269A, P271A, V2 72A, K290A, I291A, I291G, I291L, P292A, P292D, Q311A, V312A, L313A, L313P , L313M, K314A, G340Q, S341H, S341D, S341T, W342I, W342D, W342H, W342L, E 343V, E343S, E343Y, E343Q, N344F, N344I, T345E, T345K, T345S, R346M, R346 F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E349L, E349W, E349A, S350Q, S35 0I, S350D, S350T, S350E, S350N, Q352E, Q352G, Q352Y, Q352W, Q352T, A353E , A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I3 54R, I354W, I354L, K355Q, K355H, K355D, E356M, E356F, E356I, E356L, E356 Q, E356V, E356D, Y357W, Y357F, M358V, M358R, M358Y, M358L, D359K, D359V, D 359Y, D359Q, D359T, D359S, D359E, T360Y, T360R, T360L, T360D, T360S, T36 1M, T361E, T361H, T361L, T361D, T361I, L362A, N363M, N363E, L441A, S442A,The polypeptide is characterized by the substitution of one or more amino acid residues selected from the group consisting of D451A, D451S, T452A, T452D, T452H, T452K, T452G, T452P, T452M, T452F, D453A, D461R, D461A, G462A, V463Y, and V463A, but is not limited thereto.

[0047] In the present invention, an expression in which a one-letter amino acid residue name and a number are written together, such as "S341," means the amino acid residue at the corresponding position in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0048] For example, "S341" means that the amino acid residue at position 341 in the amino acid sequence of SEQ ID NO: 3 is serine, and "S341H" means that the serine at position 341 in SEQ ID NO: 3 has been replaced with histidine.

[0049] The PH20 mutant or a fragment thereof according to the present invention is understood to include mutants or fragments thereof in which amino acid residues are conservatively substituted at specific amino acid residue positions.

[0050] As used herein, "conservative substitution" refers to a modification of a PH20 variant that involves replacing one or more amino acids with amino acids having similar biochemical properties that does not result in loss of biological or biochemical function of the PH20 variant.

[0051] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains have been defined and are well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It was confirmed that the PH20 mutants or fragments thereof of the present invention can retain activity even when they have conservative amino acid substitutions.

[0052] Furthermore, the PH20 mutant or fragment thereof of the present invention is also interpreted as including a PH20 mutant or fragment thereof that has substantially the same function and / or effect as the PH20 mutant or fragment thereof of the present invention and has an amino acid sequence homology of 80% or 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.

[0053] The PH20 mutants or fragments thereof of the present invention exhibit increased expression in animal cells, increased protein refolding, and increased thermal stability compared to mature wild-type PH20, and furthermore, despite the increased thermal stability, their enzymatic activity was increased or similar to that of mature wild-type PH20.

[0054] Meanwhile, it is known that truncation of the C-terminal region of mature wild-type PH20 reduces the enzymatic activity. However, the PH20 mutant according to the present invention maintains increased or similar enzymatic activity and expression efficiency compared to mature wild-type PH20, even when some amino acid residues at the C-terminus are truncated and deleted and / or 1 to 7, preferably 1 to 5, amino acids at the N-terminus are truncated and deleted due to increased protein refolding and increased thermostability. agg )

[0055] Thus, the PH20 mutant or fragment thereof of the present invention is characterized in that, in a mutant having an amino acid sequence of the present invention, such as SEQ ID NO: 3, it contains one or more mutations, preferably substitutions, deletions and / or insertions of amino acid residues, and further contains deletions of some amino acid residues at the C-terminus and / or N-terminus, but is not limited thereto.

[0056] In one aspect, the PH20 mutant or fragment thereof of the present invention has a cleavage site before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus of the amino acid sequence of the present invention, such as SEQ ID NO: 3, preferably before an amino acid residue of L36, N37, F38, R39, A40, P41, or P42, resulting in the deletion of some amino acid residues at the N-terminus, and / or a cleavage site after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus, preferably after an amino acid residue of V456 to L509 at the C-terminus, preferably after an amino acid residue of V457 to L509 at the C-terminus. The C-terminal fragment may be characterized by a truncation occurring after an amino acid residue selected from the group consisting of V455 to S490, most preferably after the amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490, resulting in the deletion of some amino acid residues at the C-terminus.

[0057] The expression "cleavage occurs before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus" means that the amino acid residue immediately preceding the amino acid residue selected from M1 to P42 at the N-terminus has been cleaved and deleted. When cleavage occurs before M1, it means that no cleavage has occurred at the N-terminus.

[0058] For example, the expression "cleavage occurred before amino acid residue L36, N37, F38, R39, A40, P41, or P42" means that in the amino acid sequence of the present invention, such as SEQ ID NO: 3, cleavage was performed and removed from M1 to T35, the amino acid residue immediately preceding L36; from M1 to L36, the amino acid residue immediately preceding N37; from M1 to N37, the amino acid residue immediately preceding F38; from M1 to F38, the amino acid residue immediately preceding R39; from M1 to R39, the amino acid residue immediately preceding A40; from M1 to A40, the amino acid residue immediately preceding P41; and from M1 to P41, the amino acid residue immediately preceding P42.

[0059] Furthermore, the expression that cleavage occurred after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus means that cleavage began at the amino acid residue immediately following the amino acid residue selected from V455 to L509 at the C-terminus and was deleted.

[0060] For example, a statement that cleavage occurs after the C-terminal amino acid residues V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 refers to the amino acid residues of the present invention, such as SEQ ID NO: 3, respectively. In the amino acid sequence of the present invention, this means that the cleavage begins at the amino acid residue next to V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 and is removed.

[0061] Preferably, the novel PH20 mutant or fragment thereof of the present invention is characterized in that it comprises a substitution, insertion or deletion of amino acid residues at one or more positions in a mutant having the amino acid sequence of SEQ ID NO: 3, and is truncated before the N-terminal F38 residue and after the C-terminal F468 residue.

[0062] More preferably, the novel PH20 mutant or a fragment thereof according to the present invention is characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 163 to SEQ ID NO: 316, but is not limited thereto. The amino acid sequences substituted or truncated in the PH20 mutants prepared in specific examples according to the present invention are as shown in Table 6.

[0063] Furthermore, in the present invention, we attempted to improve the expression of recombinant PH20 protein by using signal peptides from other proteins that are highly expressed in animal cells, rather than the signal peptide specific to PH20.

[0064] Thus, in yet another aspect, the novel PH20 mutants of the present invention are characterized in that, instead of the signal peptide of wild-type PH20 M1 to T35, they further comprise, at the N-terminus, a signal peptide derived from human growth hormone having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA according to SEQ ID NO: 4, a signal peptide derived from human serum albumin having the amino acid sequence MKWVTFISLLFLFSSAYS according to SEQ ID NO: 5, or a signal peptide derived from human Hyal1 having the amino acid sequence MAAHLLPICALFLTLLDMAQG according to SEQ ID NO: 6, as shown in Table 3, but are not limited thereto.

[0065] The phrase "replacing the wild-type PH20 signal peptide of M1 to T35" refers to a case where the signal peptide in the amino acid sequence of SEQ ID NO: 3 is partially or completely deleted, making it unable to perform the function of the wild-type PH20 signal peptide. It also refers to a case where a portion of the N-terminus is further deleted, for example, before residues N37, F38, R39, A40, P41, or P42, resulting in a further deletion of the N-terminus along with the wild-type PH20 signal peptide.

[0066] [Table 3]

[0067] In another aspect, the present invention provides a composition for cancer treatment comprising the novel PH20 mutant or fragment of the present invention, and a method for cancer treatment using the same.

[0068] Cancers or carcinomas treatable with the novel PH20 variants or fragments of the present invention include, but are not limited to, both solid and hematological cancers. Examples of such cancers include, but are not limited to, skin cancers such as melanoma, liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, colon cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, and glioma. Preferably, cancers treatable with the compositions of the present invention are, but are not limited to, colorectal cancer, breast cancer, lung cancer, and kidney cancer.

[0069] The composition may be a pharmaceutical composition. The pharmaceutical composition may further comprise a pharmaceutically acceptable composition commonly used in drug formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. The pharmaceutical composition may also further comprise one or more commonly used diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0070] The pharmaceutical composition can be administered orally or parenterally. Parenteral administration can be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration. Since proteins or peptides are digested during oral administration, oral compositions may be coated with active agents or formulated to protect them from degradation in the stomach. The composition may be administered by any device that allows the active agent to be delivered to target cells.

[0071] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, etc., and may further contain a dispersant or stabilizer for formulation. In particular, the composition for cancer treatment according to the present invention is characterized in that it is used for combined treatment with other anti-cancer agents.

[0072] Anticancer agents that can be used in combination with the novel PH20 mutant or fragment of the present invention for therapeutic purposes include, but are not limited to, chemical anticancer agents, antibody-based anticancer agents, biological anticancer agents, RNAi, and cell therapy agents.

[0073] Preferably, the anticancer agent that can be used in combination therapy with the novel PH20 mutant or fragment of the present invention is an immune anticancer agent, particularly preferably an immune checkpoint inhibitor, but is not limited thereto. The present invention also provides a method for treating cancer, which comprises using the novel PH20 mutant or fragment of the present invention in combination with other anticancer agents, particularly the anticancer agents mentioned above. In another aspect, the present invention relates to a nucleic acid encoding the PH20 mutant of the present invention or a fragment thereof.

[0074] As used herein, a nucleic acid may be present in a cell, a cell lysate, or may be present in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" if it can be purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. Nucleic acids of the invention can be, for example, DNA or RNA.

[0075] In yet another aspect, the present invention relates to a vector comprising the nucleic acid. For expression of a PH20 variant or fragment thereof of the present invention, DNA encoding the PH20 variant or fragment thereof can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the PH20 variant), and the DNA can be "operably linked" to transcriptional and translational control sequences and inserted into an expression vector.

[0076] As used herein, the term "operably linked" can mean that a gene encoding a PH20 variant or a fragment thereof is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the gene encoding the PH20 variant or fragment thereof. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The gene encoding a PH20 variant is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the gene fragment encoding the PH20 variant or fragment thereof and the vector, or blunt end ligation if no restriction enzyme sites are present).

[0077] The recombinant expression vector also contains regulatory sequences that control the expression of a gene encoding a PH20 variant or a fragment thereof in a host cell. A "regulatory sequence" can include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of a gene encoding a PH20 variant or a fragment thereof. Those of ordinary skill in the art will recognize that the regulatory sequences can be selected differently and the expression vector can be designed differently depending on factors such as the host cell to be transformed and the desired protein expression level.

[0078] In yet another aspect, the present invention relates to a host cell containing the nucleic acid or the vector. The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells, but is not limited thereto.

[0079] Specifically, host cells according to the present invention may be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. They may also be eukaryotic cells such as fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects.

[0080] The host cells of the present invention may be derived from plants or mammals. Preferable examples include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO), W138, baby hamster kidney (BHK), MDCK, myeloma cell lines, HuT78 cells, and HEK293 cells. Particularly preferred are CHO cells.

[0081] The nucleic acid or vector is transfected into a host cell. "Transfection" or "transfection" involves introducing exogenous nucleic acid (DNA or RNA) into a prokaryotic or eukaryotic host cell using a variety of commonly used techniques, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. A variety of expression host / vector combinations may be used to express the PH20 variants or fragments thereof of the present invention. Expression vectors compatible with eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Expression vectors that can be used in bacterial hosts include bacterial plasmids derived from Escherichia coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, colE1, pCR1, pBR322, pMB9, and their derivatives; broader host range plasmids, such as RP4; phage DNA, exemplified by the wide variety of phage lambda derivatives, such as λgt10, λgt11, and NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. A useful expression vector for yeast cells is the 2 μm plasmid and its derivatives. A useful vector for insect cells is pVL941.

[0082] In yet another aspect, the present invention relates to a method for producing a PH20 mutant or a fragment thereof of the present invention, comprising the step of culturing a host cell to express the PH20 mutant or a fragment thereof of the present invention.

[0083] When a recombinant expression vector capable of expressing the PH20 variant or fragment thereof is introduced into a mammalian host cell, the PH20 variant or fragment thereof can be produced by culturing the host cell for a period of time sufficient to allow expression in the host cell, or more preferably, secretion of the PH20 variant into the culture medium in which the host cell is cultured.

[0084] In some cases, the expressed PH20 variant or a fragment thereof can be isolated from the host cells and purified to homogeneity. The isolation or purification of the PH20 variant or a fragment thereof can be carried out by a method commonly used for isolation and purification of proteins, such as chromatography. The chromatography can be, for example, a combination of one or more of affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited to these. In addition to the chromatography, filtration, ultrafiltration, salting out, dialysis, and the like can also be used in combination.

[0085] To confirm the industrial applicability of enzymes, analysis of the catalytic reaction rate of enzymes is necessary. Enzyme reactions are divided into those with an active site of fixed reactivity and those with multiple active sites with various reactivities. The catalytic reaction rate of enzymes with a single active site of fixed reactivity, such as hyaluronidase, is known to follow the Michaelis-Menten rate law.

[0086] Michaelis-Menten enzyme kinetics is based on the assumption that enzyme reactions are a two-step reaction system consisting of a reversible reaction step in the formation of an enzyme (E)-substrate (S) complex [ES], and an irreversible reaction step in which a product (P) is produced by dissociation of the ES complex, as shown in the following equation: where kf, kr, and kcat are the rate constants for the reaction in each direction (Alan Fersht (1977) Enzyme structure and mechanism).

[0087]

number

[0088] Enzyme reactions can be considered pseudo-steady-state reactions, where the enzyme and substrate react to form an ES complex and the reaction can be assumed to reach equilibrium quickly, or the reaction can be run at a high enough substrate concentration that the enzyme concentration is low enough that d[ES] / dt ≒ 0. Because the rate equations derived from either the rapid equilibrium or pseudo-steady-state reactions are identical, most experiments assume a pseudo-steady-state reaction where the substrate concentration is initially high relative to the enzyme concentration.

[0089] Under these assumptions, "the amount of enzyme is constant before and after the reaction" and "when a chemical reaction reaches chemical equilibrium, the reaction rate at which the product is produced is the same as the rate at which this substance is broken down again," the reaction rate of the final product can be expressed by the following Michaelis-Menten rate formula. In this case, K M =(k r +k cat ) / k f and V max =k cat [E]0.

[0090]

number

[0091] The Lineweaver-Burk equation is used to experimentally analyze enzyme reaction rates using the Michaelis-Menten kinetic formula. This equation shows the relationship between the reciprocal of the substrate concentration given in the experiment, 1 / [S], ​​and the experimentally measured reciprocal of the reaction rate, 1 / V. If it is statistically confirmed that this equation is a linear equation, it can be confirmed that the enzyme reaction is a reaction that follows the Michaelis-Menten kinetic formula, and this equation can be used to calculate K M and V max can be obtained.

[0092] When an enzyme catalyzes a chemical reaction, it has a transition state after binding to a substrate in the active site. The activation energy to reach the high-energy transition state is lowered by various bonds with the substrate. The equilibrium constant for reaching this transition state is k cat / K M where 1 / K M is an index that sums the degree to which the enzyme and substrate bind to form an enzyme-substrate complex and the degree to which the enzyme-substrate complex is maintained without being decomposed, and k cat is the equilibrium constant for the enzyme-substrate complex to produce the product, so k cat / K M is an index of how much product can be obtained from a substrate and an enzyme, i.e., it can be said to represent the catalytic efficiency of an enzyme.

[0093] The industrial applicability of hyaluronidase is proportional to its catalytic efficiency. In particular, when hyaluronidase is subcutaneously injected together with a macromolecular pharmacologically active substance such as a monoclonal antibody, the catalytic efficiency of hyaluronidase plays an important role. The mutants of the present invention have a higher catalytic efficiency than wild-type PH20. cat / K M When the k is high, the hyaluronidase contained in the high molecular weight pharmacologically active substance decomposes the hyaluronic acid present in the subcutaneous tissue more quickly when administered subcutaneously, thereby providing an excellent effect of enabling the pharmacologically active substance to be dispersed more quickly. cat When V is large, the maximum reaction rate at the same enzyme concentration is max As a result, a larger amount of hyaluronic acid can be decomposed in the same time period, and the pharmacologically active substance can be dispersed over a wider area, which is an excellent effect.

[0094] Therefore, to confirm the enzymatic properties of the PH20 mutants according to the present invention, the enzymatic reaction rate of each mutant was analyzed, and its V max (maximum enzyme reaction rate), K m (50% V max (substrate concentration at the condition), k cat (substrate conversion rate), k cat / K mThe results (enzyme catalytic efficiency) were compared in Example 4. These results demonstrate the superiority of the PH20 mutants of the present invention over wild-type PH20. [Example]

[0095] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not to be construed as limiting the scope of the present invention.

[0096] Example 1. Construction of PH20 mutants To generate PH20 mutants, wild-type PH20 cDNA (clone ID: hMU002604) was purchased from the Korean Human Gene Bank. Wild-type PH20 encodes amino acids L36 to S490. The PH20 gene was amplified using polymerase chain reaction (PCR) and inserted into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, the signal peptide of human growth hormone, human serum albumin, or human Hyal1 was used instead of the PH20-specific signal peptide. A His-tag DNA sequence was located at the 3' end of the PH20 cDNA for protein purification using a HisTrap column. Amino acid substitutions in the PH20 mutants were generated using PCR, and the amino acid substitutions were confirmed by DNA sequencing. The primers used for cloning the PH20 mutants are listed in Table 4, and their specific sequences are listed in Table 5.

[0097] [Table 4] TIFF0007820431000007.tif255158TIFF0007820431000008.tif255158TIFF0007820431000009.tif255158TIFF0007820431000010.tif95159

[0098] [Table 5] TIFF0007820431000012.tif244170TIFF0007820431000013.tif249170TIFF00078204310 00014.tif249170TIFF0007820431000015.tif250170TIFF0007820431000016.tif170170

[0099] After searching for PH20 mutants with increased enzymatic activity and thermostability, cDNAs of PH20 mutants were also prepared without His-tags.

[0100] The PH20 mutant was prepared using the cDNA of the PH20 mutant as follows.

[0101] The mutants were expressed using the ExpiCHO expression system. The number of ExpiCHO cells was 6 × 10 6 When the total cell count reached 100 / mL, ExpiCHO cells were transfected with a plasmid containing wild-type or mutant PH20 cDNA inserted into the pcDNA3.4-TOPO vector using ExpiFectamine CHO reagent. ExpiCHO expression medium (100-500 mL) was used for cell culture. After transfection, ExpiCHO cells were cultured with shaking at 130 rpm for a total of 6 days, including 1 day at 37°C, and then the temperature was lowered to 32°C for an additional 5 days. Upon completion of the culture, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 minutes.

[0102] The recombinant proteins of wild-type and mutant PH20 with a C-terminal His-tagged protein produced in ExpiCHO cells were purified by three-step column chromatography using an AKTA Prime system (GE Healthcare). Depending on the mutant, purification was performed using either a HisTrap HP column, a Q Sepharose column, and a phenyl HP column in that order, or a Q Sepharose column, a HisTrap HP column, and a butyl HP column in three steps.

[0103] Purification using the HisTrap HP column, Q Sepharose column, and Phenyl HP column proceeded as follows. For protein purification using the HisTrap HP column, buffer A (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 0.5 M imidazole) were prepared. Proteins were bound to the HisTrap column, and 5 column volumes of buffer A were applied to remove nonspecifically bound proteins. After confirming that the conductivity remained constant, the proteins were eluted with 5 CV of 20% buffer B. The eluted proteins were then dialyzed against dialysis buffer (20 mM sodium phosphate, pH 7.5, 50 mM NaCl). For protein purification using a Q Sepharose column, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) were prepared. Proteins were bound to the Q Sepharose column, and nonspecifically bound proteins were removed by 5 CV of buffer A. The protein was then eluted with 5 CV of buffer B in a 0-100% gradient. For protein purification using a Phenyl HP column, buffer A (20 mM sodium phosphate, pH 7.0, 1.5 M (NH4)2SO4) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared. Proteins were bound to the Phenyl column, and nonspecifically bound proteins were removed by 5 CV of buffer A. The protein was then eluted with 5 CV of buffer B in a 0-100% gradient.

[0104] Purification using a Q Sepharose column, HisTrap HP column, and Butyl HP column proceeded as follows. For protein purification using a Q Sepharose column, buffer A (20 mM NaPi, 15 mM NaCl, pH 8.0) and buffer B (20 mM NaPi, 500 mM NaCl, pH 8.0) were prepared. To make the pH and conductivity of the culture medium identical to that of buffer A, the pH was adjusted to 8 with 1 M Tris buffer, and purified water (PW) was added to adjust the conductivity to below 5 mS / cm. The culture medium was then filtered through a 0.22 μm pore membrane. After binding the protein to the next Q Sepharose column, 5 CV of buffer A was applied to remove nonspecifically bound proteins. 3 CV of buffer B was applied sequentially to elute the target protein. For protein purification using a HisTrap HP column, buffer A (20 mM NaPi, 500 mM NaCl, pH 7.5) and buffer B (20 mM NaPi, 500 mM NaCl, 500 mM imidazole, pH 7.5) were prepared. After binding of the protein sample to the HisTrap HP column, 10 CV of 7% buffer B was applied to remove nonspecifically bound proteins, followed by 3 CV of 40% buffer B to elute the target protein. For protein purification using a Butyl HP column, buffer A (20 mM NaPi, 1.5 M ammonium sulfate, pH 7.0) and buffer B (20 mM NaPi, pH 7.0) were prepared. To load the column, the protein sample was mixed with 3 M ammonium sulfate solution in a 1:1 ratio and then filtered through a 0.22 μm pore size membrane. After binding of the protein sample to the Butyl HP column, 5 CV of buffer A was applied to remove impurities. The target protein was then eluted with a linear gradient of 0-100% buffer B and dialyzed against dialysis buffer (20 mM NaPi, 100 mM NaCl, pH 7.0). The mutants according to the present invention were purified by the method described herein, and each purified product was subjected to 10% SDS-PAGE analysis, as shown in Figures 1 and 3. The enzyme activity of wild-type and mutant PH20 was measured using a turbidimetric assay.

[0105] Turbidimetric analysis is a method for measuring the absorbance of precipitates formed when hyaluronic acid is mixed with albumin (BSA). When hyaluronic acid is hydrolyzed by PH20, the absorbance decreases when mixed with albumin. The general procedure is as follows: Hyaluronidase PH20 (Sigma) is diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in individual tubes. The purified protein sample is dissolved in enzyme diluent buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and diluted to 100X, 300X, 600X, 1200X, and 2400X. In a new tube, dilute the 3 mg / mL hyaluronic acid solution 10-fold to a concentration of 0.3 mg / mL, making each tube a 180 μL volume. Add 60 μL of enzyme to the diluted hyaluronic acid solution, mix, and react at 37°C for 45 minutes. After the reaction is complete, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate, shake for 10 minutes, and then measure the absorbance at 600 nm using a spectrophotometer.

[0106] Protein thermal stability was measured by measuring the aggregation temperature using a dynamic light scattering (DLS) instrument, and the melting temperature (T) measured by real-time PCR using Sypro Orange dye. m ), or measuring enzyme activity after leaving it at a specific temperature for a certain period of time. DLS aggregation temperature measurement is highly sensitive because it uses light scattering to measure the aggregation phenomenon of molecules, and is generally used to measure the melting point (T m) is lower than the temperature. Each mutant is prepared in the same solution of 0.2 mg / mL and then measured, and this result can be used as the aggregation temperature to compare the physical properties of each mutant (Philo, JS (2009) Cur Pharm Biotech 10,359-372). The amino acid sequences of the PH20 variants prepared in the present invention by substitution or truncation from the PH20 variant of SEQ ID NO: 3 are shown in Table 6 below.

[0107] In the present invention, experiments were performed using mutants with six histidines added to the C-terminus of the sequence shown in Table 6 for protein purification, and it was confirmed that this C-terminal addition did not affect the enzyme activity or protein stability. The mutants according to the present invention are named by a combination of HM and a sequence number, and the mutants according to Example 3 are named Hyal2 mutant, Hyal3 mutant, and Hyal4 mutant, respectively.

[0108] [Table 6] TIFF0007820431000018.tif220170TIFF0007820431000019.tif201170TIFF0007820431000020.tif201170TIFF0007820431000021.tif201170TIFF0007820431000022.tif201170TIFF0007820431000023.tif201170TIFF0007820431000024.tif201170TIFF0007820431000025.tif201170TIFF0007820431000026.tif201170TIFF0007820431000027.tif201170TIFF0007820431000028.tif201170TIFF0007820431000029.tif201170TIFF0007820431000030.tif201170TIFF0007820431000031.tif201170TIFF0007820431000032.tif201170TIFF0007820431000033.tif201170TIFF0007820431000034.tif201170TIFF0007820431000035.tif201170TIFF0007820431000036.tif201170TIFF0007820431000037.tif201170TIFF0007820431000038.tif201170TIFF0007820431000039.tif201170TIFF0007820431000040.tif201170TIFF0007820431000041.tif201170TIFF0007820431000042.tif201170TIFF0007820431000043.tif201170TIFF0007820431000044.tif201170TIFF0007820431000045.tif201170TIFF0007820431000046.tif201170TIFF0007820431000047.tif201170TIFF0007820431000048.tif201170TIFF0007820431000049.tif201170TIFF0007820431000050.tif201170TIFF0007820431000051.tif201170TIFF0007820431000052.tif20117 0TIFF0007820431000053.tif201170TIFF0007820431000054.tif201170TIFF0007 820431000055.tif201170TIFF0007820431000056.tif201170TIFF0007820431000 057.tif201170TIFF0007820431000058.tif201170TIFF0007820431000059.tif20 1170TIFF0007820431000060.tif201170TIFF0007820431000061.tif201170TIFF 0007820431000062.tif201170TIFF0007820431000063.tif201170TIFF000782043 1000064.tif201170TIFF0007820431000065.tif201170TIFF0007820431000066.t if201170TIFF0007820431000067.tif201170TIFF0007820431000068.tif176170.

[0109] Example 2. Characterization of PH20 mutants according to the present invention Further studies on the structure and function of the protein were carried out through mutation studies, including truncation of the N- and C-terminus, based on the amino acid sequence of SEQ ID NO: 3. The expression levels and activity of the mutants produced were analyzed, and the aggregation temperatures were as shown in Table 7.

[0110] Expression level and specific activity were measured using the turbidimetric assay described in Example 1. When summarizing the results of this assay, limits of quantification (LOQ) were set for the culture medium and purified activity assays, respectively. When the culture medium activity exceeded 300 units / mL, it was indicated as ">LOQ." When the activity exceeded 15 units / μg, it was indicated as ">LOQ." In the opposite cases, the inequality sign was changed. In Table 7, this was applied to indicate the quantification limits for each expression level and activity assay, and the test results were summarized based on this. The aggregation temperature of wild-type PH20 (L36-Y482) of SEQ ID NO: 1 was 46.5°C, and the aggregation temperature of the PH20 mutant (F38-F468) of SEQ ID NO: 3 was 51°C.

[0111] [Table 7] TIFF0007820431000070.tif255167TIFF0007820431000071.tif255167TIFF00078204310 00072.tif255167TIFF0007820431000073.tif249167TIFF0007820431000074.tif194167

[0112] As shown in Table 7, the mutants having the amino acid sequence of SEQ ID NO: 3 and having a substitution of one amino acid residue are HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM97, HM98, HM99, and HM100. , HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM1 30, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144, HM145, HM152, HM153, HM154, HM155, HM 156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196 A total of 133 mutants, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245, and HM246, were confirmed to maintain activity in the purified fractions after purification and to be highly thermostable mutants with aggregation temperatures ranging from 48°C to 58°C.Of these, HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM98, HM99, HM100, HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, A total of 65 mutants, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144, and HM145, are mutants in which one of the substitution sites in SEQ ID NO: 3 that was substituted with PH20 has been mutated, and their aggregation temperatures range from 48°C to 58°C. HM97, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM1 71, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194 A total of 68 mutants, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245, and HM246, are mutants in which a mutation has been made at one position other than the substitution site substituted from PH20 in SEQ ID NO: 3, and their aggregation temperatures range from 48°C to 56°C.

[0113] This indicates that mutants with a single substitution in SEQ ID NO: 3 have a higher aggregation temperature than the wild-type PH20 (L36-Y482) of SEQ ID NO: 1, regardless of the substitution position. However, among these, HM174, HM208, HM210, and HM211 were found to have activities in the culture medium lower than the LOQ of 300 units / mL, but their activities after purification were higher than the LOQ of 15 units / μg. From these examples, it can be concluded that measuring the activity of mutants only in the culture medium makes it difficult to accurately analyze the characteristics of the mutants themselves.

[0114] Furthermore, as shown in Table 7, among the mutants having the amino acid sequence of SEQ ID NO: 3, HM146, HM147, HM149, HM262, and HM263 maintain the same mutations, i.e., amino acid residue substitutions, as the mutant having the amino acid sequence of SEQ ID NO: 3, but include additional truncations at the N- and C-termini, confirming that the additional truncations at the N- and C-termini do not affect the expression and activity of the protein in the mutants having the amino acid sequence of SEQ ID NO: 3. The aggregation temperature also remains in the range of 49°C to 53°C, which is not significantly different from that of SEQ ID NO: 3, indicating that the additional truncations at the N- and C-termini do not affect the physical properties.

[0115] Furthermore, among the variants having the amino acid sequence of SEQ ID NO: 3, 13 variants containing one or more amino acid substitutions and truncations, including HM96, HM150, HM254, HM261, HM266, HM268, HM271, HM275, HM276, HM279, HM280, HM287, and HM288, were also successfully expressed and maintained their enzymatic activity, with aggregation temperatures ranging from 48°C to 59°C. Therefore, even in the case of multiple substitutions, the activity and physical properties of the protein were maintained. However, in the case of multiple substitutions, the enzymatic activity and aggregation temperature were observed to be unpredictable based solely on the combination of properties exhibited by the individual single substitutions.

[0116] Example 3. Activity analysis of mutants substituted with sequences of Hyal2, Hyal3, and Hyal4 In place of the M345-I361 section of the amino acid sequence of wild-type PH20 of SEQ ID NO: 1, the amino acid sequences of Hyal2 (TTSTETCQYLKDYLTRL), Hyal3 (SSSEEECWHLHDYLVDT), and Hyal4 (TASKANCTKVKQFVSSD), which are other types of hyaluronidases present in humans in addition to Hyal1, were introduced into the M345-I361 section, and the changes in protein stability were examined.

[0117] Mutants in which the M345 to I361 sites of mature wild-type PH20 (L36-S490) were replaced with the corresponding sequences of Hyal2, Hyal3, and Hyal4, respectively, are referred to as Hyal2 mutant, Hyal3 mutant, and Hyal4 mutant, respectively.

[0118] After generating Hyal2, Hyal3, and Hyal4 mutants, we analyzed their thermal stability (see Figure 2). As a result, the aggregation temperature of the Hyal3 mutant measured by DLS was 48°C, which was 1.5°C higher than that of wild-type PH20 (46.5°C), indicating increased thermal stability.

[0119] To confirm the expression of these mutants in ExpiCHO cell cultures, the mutants were purified using the same method using a HisTrap column, and the protein expression levels were compared by SDS-PAGE analysis. The Hyal3 mutant was found to be most highly expressed, followed by the Hyal2 and Hyal4 mutants.

[0120] Example 4. Thermostability analysis of mutants according to the present invention To confirm the thermal stability of the mutants of the present invention, SDS-PAGE analysis was performed. Purified wild-type PH20 (L36-Y482) of SEQ ID NO: 1 and the purified protein (F38-F468) of the PH20 mutant of the present invention of SEQ ID NO: 3 were stored at 42°C for 7 days, and then subjected to 10% SDS-PAGE analysis under reducing and non-reducing conditions (Figure 4).

[0121] As a result, aggregates were observed in wild-type PH20 (L36-Y482) (lane G in Figure 4), but no aggregates were observed in the PH20 mutant of SEQ ID NO: 3 (F38-F468) (lane H in Figure 4). This confirmed that the difference in aggregation phenomenon was due to the difference in aggregation temperature between the two proteins. Therefore, due to the high aggregation temperature of the mutant of the present invention, it is believed that it has higher thermal stability than wild-type PH20 and may be more widely applicable industrially.

[0122] Example 5. Enzyme kinetics analysis of the mutants of the present invention To analyze the enzyme kinetics of the mutants of the present invention, enzyme activity was measured by the Morgan-Elson method (Takahashi, T. et al. (2003) Anal Biochem 322:257-263). The Morgan-Elson method is a colorimetric assay that quantifies (at 545 nm) the red substance produced by the reaction of the reducing end of N-acetyl-D-glucosamine (GlcNAc) produced when hyaluronic acid is hydrolyzed by hyaluronidase with the Ehrlich's reagent, DMAB (para-dimetylaminobenzaldehyde). N-acetyl-D-glucosamine (GlcNAc, Sigma) was diluted with dilution buffer (0.1 M NaPi, 0.1 M NaCl, 1.5 mM saccharinic acid 1,4-lactone, pH 5.35) to 0.25, 0.50, 0.75, 1.00, and 1.25 mM. The solution was treated with tetraborate and reduced, followed by the addition of DMAB for color development. Absorbance was measured at 545 nm to generate a standard reaction curve for GlcNAc. Hyaluronic acid, the substrate, was diluted with dilution buffer to 0.54, 0.65, 0.87, 1.23, and 2.17 μM. Hyaluronidase was added to each test tube. The reaction was then incubated at 37°C for 5 minutes, followed by heating at 100°C for 5 minutes to terminate the enzyme reaction. The solution was then treated with tetraborate and reduced, followed by the addition of DMAB for color development. After the reaction, absorbance was measured at 545 nm, and enzyme activity was measured using the standard reaction curve for GlcNAc. When the enzyme kinetics of wild-type PH20 (SEQ ID NO: 1) and the PH20 mutants of the present invention were analyzed using this method, the linearity of the Lineweaver-Burk curves was confirmed, confirming that the PH20 mutants of the present invention follow the Michaelis-Menten enzyme kinetic equation.

[0123] Table 8 shows the enzyme kinetic analysis results for wild-type PH20 (L36-Y482) of SEQ ID NO: 1, PH20 mutant (F38-F468) of SEQ ID NO: 3, HM261, and HM268. max(maximum enzyme reaction rate), K m (50% V max (substrate concentration at the condition), k cat (substrate conversion rate), k cat / K m (enzyme catalytic efficiency) was presented. m The smaller the value, the greater the enzyme's substrate binding strength, and k cat The larger the value, the greater the enzyme's substrate conversion rate, so k cat / K m It was confirmed that the enzyme catalytic efficiency of each PH20 mutant was increased compared to that of wild-type PH20. cat Since the values ​​are higher than those of the wild-type PH20 of SEQ ID NO: 1, the substrate conversion rate of the enzyme is higher, and therefore, the industrial applicability of each PH20 mutant is greater than that of the wild-type PH20.

[0124] [Table 8]

Claims

1. A PH20 mutant, The amino acid sequence of the PH20 mutant is (a) (i) amino acid residue substitutions T132S, L181A, E186D, Q268N, I291L, V312A, and T452D relative to the amino acid sequence of SEQ ID NO: 3; (ii) amino acid residue substitutions R39K, I105A, T132S, L181M, E186D, Q268A, I291L, V312A, and T452D relative to the amino acid sequence of SEQ ID NO:3; (iii) amino acid residue substitutions T132A, L181A, E186A, Q268A, I291L, V312A, and T452D relative to the amino acid sequence of SEQ ID NO: 3; (iv) amino acid residue substitutions T132S, L181A, E186D, Q268N, I291L, V312A, K348M, and T452D relative to the amino acid sequence of SEQ ID NO: 3; (v) amino acid residue substitutions T132S, L181A, E186D, Q268N, I291L, V312A, N344I, K348M, and T452D relative to the amino acid sequence of SEQ ID NO: 3; (vi) amino acid residue substitutions T132A, L181A, E186A, Q268A, I291L, V312A, S341D, W342L, E343S, N344I, T345S, R346S, K348M, K355D, D359E, T361I, and T452D relative to the amino acid sequence of SEQ ID NO: 3; or (vii) amino acid residue substitutions T132A, L181A, E186A, Q268A, I291L, V312A, S341D, W342L, E343S, N344I, T345S, K348M, K355D, D359E, T361I, and T452D relative to the amino acid sequence of SEQ ID NO: 3; and (b) an N-terminal deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, or M1 to R39 of SEQ ID NO: 3; and (c) a C-terminal deletion, wherein the C-terminus of the PH20 variant ends with any one of amino acid residues V455 to S490 of SEQ ID NO: 3; and The PH20 variant has an aggregation temperature of at least 48°C, and The PH20 variant has hyaluronidase activity. PH20 mutant.

2. A PH20 mutant described in claim 1, wherein the N-terminal deletion is a deletion of amino acid residues M1 to N37 of SEQ ID NO: 3, and the C-terminus of the PH20 mutant ends at amino acid residue F468 of SEQ ID NO:

3.

3. A PH20 mutant described in claim 1, wherein the amino acid sequence of the PH20 mutant consists of any one of the amino acid sequences of SEQ ID NOs: 305, 308, 309, 313, 314, 315, and 316.

4. A PH20 mutant described in claim 1, wherein hyaluronidase activity is measured by turbidimetry.

5. A composition for use in cancer treatment, comprising a PH20 mutant according to any one of claims 1 to 4.

6. The composition according to claim 5, which is used in combination therapy with other anticancer agents.

7. The composition of claim 6 , wherein the other anti-cancer agent is an immunological anti-cancer agent.

8. The composition of claim 7 , wherein the immune anti-cancer agent comprises an immune checkpoint inhibitor.

9. A nucleic acid encoding the PH20 mutant of any one of claims 1 to 4.

10. A recombinant expression vector comprising the nucleic acid of claim 9.

11. A host cell transformed or transduced with the recombinant expression vector of claim 10.

12. A method for producing a PH20 variant, comprising culturing a host cell according to claim 11.

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